Phosphorylation of the estradiol receptor in MCF-7 human breast cancer cells in culture.
Double labelling and Western blot techniques were used to demonstrate phosphorylation of estradiol receptor. Cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine [( 3H]TA). Labelled receptor was precipitated with the monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or transferred to nitrocellulose paper. Receptor protein was detected on the Western blot with the monoclonal antibody H222 and rabbit anti-rat peroxidase conjugate. Phosphorylated receptor was visualized by autoradiography. Tritium and 32P activities were monitored in the gels. Two phosphorylated forms of the receptor (molecular weights 67 and 50 kDa) have been detected in MCF-7 cells. Estradiol treatment of the cells was found to increase phosphorylation of the receptor. In estradiol-treated cells both phosphorylated receptor forms were present mainly in the nuclear extract. Both forms bound [3H]TA as evidence by SDS-PAGE. [3H]TA binding was abolished by excess non-radioactive estradiol. In addition two phosphorylated proteins of approximately 120 and 90 kDa were regularly coprecipitated with receptor in cytosol. These proteins did not bind [3H]TA. The 90 kDa phosphorylated protein was identified as a heat shock protein (hsp-90).
Lahooti
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H
Department of Biochemical Endocrinology, University of Bergen, Haukeland Hospital, Norway.
Thorsen
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T
Aakvaag
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A
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D011960:Receptors, Estrogen / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; D013629:Tamoxifen; D004958:Estradiol; C031859:tamoxifen aziridine